Medicinal plants used in traditional treatment of malaria in Ethiopia: a review of ethnomedicine, anti-malarial and toxicity studies

Background Malaria is extremely common in Ethiopia, and it is one of the country's most serious public health and economic problems. Traditional medicines have long been utilized in Ethiopia by people of various ethnic groups. As a result, the goal of this study is to record the use of Ethiopian medicinal herbs that have been used to treat malaria. Also, a critical review of the literature on the therapeutic properties of these and other Ethiopian medicinal plants that have been tested against Plasmodium spp. parasites was conducted with the goal of highlighting neglected studies and fostering further research in this area. Methods A comprehensive literature search was performed in Scopus, Web of Science Core Collection, PubMed, Science Direct, Google Scholar, and Scientific Electronic Library Online (SciELO) from August 2021 to October 2021. The study databases included original articles published in peer reviewed journals covering anti-malarial plants, dated until October 2021. Results The review looked at 51 plant species (28 families) that have been used to treat malaria in Ethiopia. The most often used ethnobotanical plant species for the treatment of malaria were Allium sativum, Croton macrostachyus, Carica papaya, and Lepidium sativum. Leaves were used more frequently as a therapeutic preparation than other parts. Plant extracts were found to have very good, good, and moderate anti-malarial activity in mice with rodent Plasmodium species. The most active species were Ajuga remota and Capsicum frufescens, which suppressed parasitaemia by 77.34% and 72.65%, respectively, at an oral dose of 100 mg/kg and an LD50 of above 2000 mg/kg. The compound Aloinoside reported from Aloe macrocarpa leave latex was the most potent; it suppressed parasitaemia by 100% at 400 mg/kg oral dose of Plasmodium berghei infected mice, and its LD50 was above 2000 mg/kg. Toxicity was shown to be safe in 84% of the plant extracts. Conclusion In Ethiopia, medicinal plants have a significant part in reducing the severity of malaria due to their widespread use. As a result, more studies are needed to identify and develop effective novel drugs that could be employed in broader malaria eradication efforts.

estimated 3.3 billion people globally [2], and it is a major public health issue in tropical and subtropical areas [3]. According to the World Health Organization (WHO), 229 million new cases of malaria were reported worldwide in 2019, and an estimated 409,000 people died from malaria in that period. The majority of malaria cases and resulting mortality occurred in the WHO African area (94%) [4]. Malaria causes major complications in infected people, such as severe anaemia, cerebral malaria, acute renal failure, and hypoglycaemia [5]. Five Plasmodium species are responsible for the disease [6] and four of these species occur in Ethiopia-Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae [7].
Plasmodium falciparum is the most severe Plasmodium species in terms of morbidity and mortality, followed by Plasmodium vivax with proportions of 60% and 40%, respectively [8]. Malaria is one of Ethiopia's most serious public health and economic issues. The prevalence of malaria in children and pregnant women are 0.6% and 16.3%, respectively [9,10]. In Ethiopia, the transmission patterns and intensity vary greatly due to the large diversity in altitude, rainfall, and population movement, with areas below 2000 m being potentially malarious. Those areas are home to approximately 68% (52 million) of the Ethiopian population and cover almost 75% of the country's landmass, resulting in around 10 million clinical cases each year according to Ethiopian National Malaria Indicator survey of 2007 [11]. Ethiopia is one of the countries that have adopted the revised malaria control strategies. The most crucial in malaria prevention and control strategies are indoor residual spraying and long-lasting insecticidal nets. In Ethiopia, quick diagnostic tests are being introduced at the community level, as well as the adoption of artemisinin-based combination therapy (ACT). However, there have already been instances of increased treatment failure and probable resistance to certain combinations [12][13][14]. As a result, new medications as prospective substitutes for artemisinin-based combinations are urgently needed. Pharmaceutical firms, on the other hand, consider a large investment in the development of new (semi)synthetic anti-malarial medications to be a dangerous venture, because the populations of developing nations cannot afford to pay a high price for these drugs. There is a need to develop new cost effective anti-malarial drugs to assist in controlling malaria and reducing its impact in these areas until eradication programmes become realistic.
One approach to the development of novel anti-malarial drugs is to reinvestigate traditional medicines. In this context, Ethiopia possesses a diverse range of medicinal plants linked to a variety of traditional medical practices that vary by ethnic group [15]. Despite this, there is a paucity of well-documented ethnobotanical and ethnopharmacological literature on Ethiopian anti-malarial herbs. The review looked at the various ethnomedicinal studies that have been conducted, as well as the scientific validation of antiplasmodial activity, anti-malarial activity, toxicity, and phytochemistry of these plants utilized in Ethiopian traditional medicine. This review may open the way for additional supplementary research as well as the development of a number of readily available and affordable anti-malarial phytomedicines, in keeping with the goals of the WHO's "Traditional Medicine Strategy" [16]. Articles offering information on malaria or medicinal plants in Ethiopia were given utmost priority throughout all publishing years. As a result, references found in the returned results were evaluated for inclusion in this study, and further searches were conducted using more general search terms such as "Ethiopian," "medicinal plant," "traditional medicine," "ethnomedicine," "parasite," "malaria," "anti-malarial," and "antiplasmodial". The study was non-biased, with no preference for endemic species or taxonomic preference. The initial ethnobotanical literature search did not include scientific evidence to support traditional use, but it was added in subsequent searches to see if the traditional use had been validated. The search was restricted to studies that were written in English. Relevant articles were identified and the data extracted by the reviewers: plant species, plant family, parts of the plant used, methods of preparation, type of study (whether in vitro or in vivo), the extraction solvent used, IC 50 or ED 50 values, parasite suppression rate, isolated compounds, strain of Plasmodium tested and toxicity.

Categorization of anti-malarial and antiplasmodial activities
For in vitro investigations, antiplasmodial activity of extracts was rated very good if the IC 50 was less than 5 μg/ml, good if the IC 50 was greater than 5 μg/ml and less than 10 μg/ml, and moderate if the IC 50 was 10 μg/ ml ≤ IC 50 < 20 μg/ml [17]. For in vivo investigations, an extract's anti-malarial activity is deemed very good if it suppresses malaria by ≥ 50% at 100 mg/kg body weight/

Bulbs
The bulb, which is free of external scales, is crushed and blended with honey before being consumed on an empty stomach [26] Bulbs Allium sativum bulb and Ginger officinale rhizome are pounded and eaten with honey [27] Fruits Fresh or dry fruits is chewed orally [28] Bulbs Before eating breakfast, take the bulb with Ethiopian traditional food 'injera' and Capsicum annuum for 5 days [18] Fruits Crush the fruit and boil it, then drink it with much amount of milk for 1 day [29] Bulbs Crush it and consume it alone or mixed with Lepidium sativum seeds [30] Allium cepa Keye shinkurt (A) Bulbs Chew the bulbs and swallow it [31] Aloaceae ( Aloe megalacantha Ere (T) Leaves Crush the leaves to get the juice, then filter and drink the filtrate [30] Asteraceae (5) Artemisia abyssinica Aritimiza (Had) Leaves Fresh leaves were crushed and pounded with water, filtered and drunk until they were recovered [34] Vernonia amygdalina Grawa (A) Leaves and barks For days, morning and evening, leaves and bark mixed with honey are consumed [35] Leaves Crushed leaves of Vernonia amygdalina concocted with leaves of Ruta chalepensis. One cup is served as a drink for 3-5 days with cold water in the morning [22] Artemisia afra Chugughee (A) Leaves Powdered fresh/dry leaves mixed with butter is taken with coffee orally before breakfast for three days [28] Leaves Fresh leaves crushed and pounded with water and then filtered and drunk in one tea cup [36] Calpurnia aurea Digita (A) Leaves Maceration, taken orally once daily for seven days [37] Echnops kebericho Kebericho (A) Roots Maceration; take orally once daily for seven days [37] Boraginaceae (1) Cordia africana Wanza (A) Roots and Barks Decoction of roots and inner bark with ginger is consumed [35] Brassicaceae (1) Lepidium sativum Feto (A) Fruits Dried fruit is ground into powder, mixed with castor oil, and administered orally [35] Seeds Pounded seeds mixed with Allium sativum bulbs and honey is taken orally for five days before breakfast After each dose, one glass of melted butter is recommended for immediate recovery [28] Seeds The seed is powdered and made as Porridge with other grains [38] Capparidaceae (1) Maerua oblongifolia Ja"a (O) Leaves Pounded leaves boiled with goat milk and drunk. It is also taken in mixture with the leaves of Withania somnifera [33] Caricaceae (1) Carica papaya Papaya (A) Leaves Squeezed the fresh leaves juice and drunk [39] Leaves The fresh leaves crush and drink with milk or without milk [36]

Leaves
When the leaves become yellow, that means getting to dry, powdered and boiled in water and a cup of tea will be taken for 5 days [18] Leaves Leaves are pounded and then boiled; the decoction is taken while cold [38] Caryophyllaceae (1) Silene macrosolen Saerosaero (T) Roots Crush and place it on fire for fumigation [30] Combretaceae ( Leaves Crushing leaves and drinks with either Guizotia abyssinica or milk [29] Euphorbia abyssinica Kulkual (A) Latexs Fresh latex of Euphorbia abyssinica eat bake with Eragrostis tef dough [26] Jatropha curcas Habet-muluk (So) Leaves The outer cover of the seed removed and the inside part swallowed with camel milk or chewed [33] Euphorbia abyssinica Kulkual (A) Roots Crushing the root and drink with milk [29] Fabaceae (9) Sesamum indcum Eshkulubia (Ku) Roots Pounding the fresh root, mixed with boiled milk and drink a half cup of it in the morning and afternoon [40] Tephrosia gracilipes Atotoka (Ku) Roots Crushing the dried root, homogenize with water and drink a bottle cup of it in the morning in empty stomach [40] Acacia seyal Tundukiyac (O) Barks Gum from bark is chewed [35] Albizia amara Ondoddee (O) Barks Bark is chewed [35] Tamarindus indica Mala (B) Fruits Chopped, dispersed in water and the suspension is drunk [42] Cicer arietinum Shinbira (A) Seeds The dried seeds germinate, then eat them with an Allium sativum bulb [26] Entada abyssinica The bark ground along with rhizome of Zingiber officinale and bulb of Allium sativum and chewed once a day for few months [22] Tamarindus indica Ged-Kinin (So) Fruit/pulp Infusion of the fruit/pulp kept overnight and drunk after taking goat soup [33] Senna italica Salamaki (So) Leaves Dried leaves powdered and boiled with water and drunk after adding goat or camel milk [33] Flacourtiaceae (1)

Ajuga integrifolia Anamuro (Hal) Leaves
The fresh whole parts of plant crushed, the liquid is filtered & drunk it [32] day, good if it suppresses malaria by ≥ 50% at 250 mg/kg body weight/day, and moderate if it suppresses malaria by ≥ 50% at 500 mg/kg body weight/day [17].

Ethiopian medicinal plants used traditionally to treat malaria
Ethiopia has a diverse flora, and some local people employ several of the plant species for medical purposes [18]. The widespread use of traditional medicines in Ethiopia can be linked to its cultural acceptability, efficacy against specific ailments, physical accessibility, and economic affordability when compared to modern medicine [19]. Traditional remedies are the most important and, in some cases, the only source of treatments for approximately 80% of Ethiopians, and 95% of the preparations are made from plants [19]. In different locations of Ethiopia, 51 plant species from 28 families were reported as being engaged in the treatment of malaria ( Table 1). The following families account for 64% of the anti-malarial plant species documented across the country: Fabaceae has nine species, Asteraceae has five, Euphorbiaceae has four, Aloaceae has three, Solanaceae has three, Alliaceae has two, Urticaceae has two, and Meliaceae has two species. Allium sativum, Croton macrostachyus, Carica papaya, and Lepidium sativum were the most commonly employed ethnobotanical plant species for the treatment of malaria (Table 1). The use of plant components and the manner in which they are prepared are limited by their availability and indigenous people's expertise [20]. According to the results of the analysis of the plant parts used, traditional healers used the leaves the most, accounting for around 42% of the total plant parts used (Fig. 1). Damage to medicinal plants caused by leaves harvesting is negligible when compared to other components [21]. The majority of these anti-malarial botanicals are employed as monotherapies, but others are utilized in combination therapies. The combination of Entada abyssinica barks, Zingiber officinale rhizomes, and Allium sativum bulbs chewed once a day for a few months to treat malaria is an example of a multi-herbal combination [22].
According to the review, crushing, powdering, macerating and decoction were the most common remedy formulations, and oral administration was the most common mechanism of route of delivery. In the preparation of remedies, water and various ingredients such as honey, butter, salt, coffee, tea, and milk were frequently employed. It was believed that the additions would either reduce toxicity or increase flavor. A variety of plant species used to treat malaria in Ethiopia has also been found in other African countries. Anti-malarial such as Rumex abyssinicus and Vernonia amygdalina have been utilized in Kenya [23], Carica papaya and Azadirachta indica have been used in Southwest Nigeria [24]. Many similar examples exist amongst those documented in Table 1.

Scientific studies into the anti-malarial activity of Ethiopian medicinal plants
In vivo anti-malarial properties of extracts from 38 plant species studied in mice with rodent Plasmodium species were indicated for the treatment and/or prevention of malaria (Table 2). Seven studies (16%) indicated very good activity (suppression rate of 50% at 100 mg/kg body weight/day), sixteen studies (37%) reported good activity, and twenty studies (47%) found moderate activity. All in vivo experiments have been conducted using the 4-day suppressive test [47] and the Rane (curative) test [48]. Aloaceae and Asphodelacea were the most studied plant families. It is possible that this is an account of the Aloaceae family, which is found in every floristic region of the country [49]. Aerial parts, leaves, leaf latex, rhizomes, roots, stem bark, fruits, and seeds were among the plant parts examined for anti-malarial activity. The crude extract of the plants was employed in the majority of the studies (76%). Acanthus polystachyus [50,51], Aloe debrana [52,53], Combretum molle [54,55], Croton macrostachyus [56,57], Clerodendrum Myricoides [53,58] and Dodonaea angustifolia [53,59,60] are examples of plant species that have been studied by more than one author. Echinops kebericho, Artemisia abyssinica, Aloe megalacantha, Carica papaya, Combretum molle, Croton macrostachyus, Ajuga remota, and Dodonaea angustifolia are some of the plant species listed in Table 2 that have anti-malarial activity, which supports the traditional uses indicated in Table 1. In experiments using a methanolic extract of the leaves, the most active species was Ajuga remota (' Akorarach' in Amharic; 'Etse-Libawit' in Ge'ez), which provided a high suppression of parasitaemia of 77.34% with an oral dose of 100 mg/kg [61]. Despite the fact that it is often suggested that more polar solvents such as water, methanol, and ethanol be used only in traditional preparations [62]. Surprisingly, the anti-malarial activity of most of the plant species studied matched to high polarity (methanol) plant extracts in most studies. According to Lipinski's laws of 5 [63], this is beneficial because it allows therapeutic components to absorb through the gut lumen into the circulatory system, where they are needed. As a result, active compounds work through cell surface receptors, with polar components providing clinically relevant potency in vivo.

Toxicity of plants extract evaluated for their anti-malarial activity
In oral acute toxicity evaluation of the test extract, 36 studies studied toxicity assays out of 43 in vivo studies (Table 2), and 84% were found to be mortality or symptoms of toxicity was not observed, which could explain the plant's safe for folkloric use. In comparison to an in vitro investigation, the in vivo model was chosen    10-Hydroxy-10 (chrysophanol-7'-yl)chrysophanol anthrone (5) 1.7 and 0.7 μg/ml 10-Methoxy-10-(chrysophanol-7'-yl)chrysophanol anthrone (6) 4 Aloesaponarin I (19) 5.0 μg/ml because it takes into consideration any pro-drug effect and the likelihood of the immune system managing infection [53]. The leaves were the plant part that had the most toxicity reports. Toxicity tests have indicated that several plant species with various parts, such as Combretum molle stem barks and seeds [54,55] and Croton macrostachyus leaves, fruits, and roots [56,57], are harmless.

Reported compounds characterized as anti-malarial and antiplasmodial in Ethiopian medicinal plants
Ten plant species used in Ethiopian folkmedicine for malaria treatment have been shown to contain antimalarial and antiplasmodial active compounds. The majority of the active compounds reported are anthraquinones, followed by naphthalene derivatives. Alkaloids are one of the most common types of compounds with anti-malarial activity. However, many naturally occurring nonalkaloidal compounds, such as terpenes, limonoids, chromones, xanthones, flavonoids, and anthraquinones, have anti-malarial activity when tested in various malarial models, according to a number of studies [93]. 14 (61%) of the reported compounds have been examined in vivo, whereas 9 (39%) have been examined in vitro against P. falciparum. There were 56% of very good, 35% of good, and 9% of moderate activity among the compounds reported. Details about these bioactive compounds are given below as well as in Table 3 and Fig. 2. The in vivo studies done by Melaku et al. [60], showed that three known compounds pinocembrin (20), flavanol santin (21) and clerodane diterpene 2-hydroxy-15, 16-epoxyceloda-3, 13, 14-trien-18-oic acid (22) were reported from Dodonaea angustifolia leaves and bio-assayed for their anti-malarial activities against Plasmodium berghei. According to the findings, compounds exhibited significant percent suppression of parasitaemia by 81% at 40 mg/kg, 80% at 50 mg/kg and 70% at 40 mg/kg, respectively in mice infected with P. berghei. Aloinoside (17) was reported from Aloe macrocarpa leave latex and evaluated for anti-malarial activity; it suppressed parasitemia by 100% at 400 mg/kg oral dose in P. berghei infected mice, and its LD 50 was above 2000 mg/kg [73]. This suggests that this compound could be employed as an anti-malarial drug. Other phytochemicals, Aloin (16) reported in the latex of Aloe debrana leaves latex, inhibited infection by 78.3% at 100 mg/ kg body weight and increased the survival time of mice infected with P. berghei [52].
In the studies with Otostegia integrifolia, very low doses of Otostegindiol (14), the active principle (25, 50, 100 mg/ kg body weight), have been tested, resulting in chemosuppression of 50.13, 65.58 and 73.16%, respectively, in P. berghei (strain ANKA)-infected mice [94]. Because such low doses are clinically feasible for human use, efforts should be focused on the development of anti-malarial compounds with higher activity at low doses. However, because certain natural products are metabolized and the pharmacokinetics of individual natural products are frequently ignored, the likelihood that the in vitro data given (Table 3) in studies with some phytochemicals may be misleading cannot be overlooked. Compounds that are said to be active in vitro may be inactive in vivo. More pharmacokinetic studies using these phytochemicals would be tremendously beneficial, though it should be noted that most of the time, small quantities of these compounds are isolated, which limits in vivo studies. Moreover, some of the phytochemicals which have been reported to be active in vivo, exhibited such activities only at very high doses that may not have meaningful therapeutic use. Also, the toxicity of almost all of these purified compounds have not been be evaluated. This severely limits their potential as anti-malarial drugs in the future. Considering the importance of cytotoxicity tests, the selectivity index for all plant extracts (Table 2) and purified compounds (Table 3) has yet to be computed. The significance of the SI (CC 50 [95]. All these have brought limitations on some of the reported compounds being considered as lead molecules for anti-malarial drug development. Therefore, the purified compounds must be further investigated, taking into account the limitations in the development of new antimalarial drugs and/or indicating the best anti-malarial remedies.

Conclusion
As a result of several ethnobotanical investigations conducted in Ethiopia, a great variety of plants utilized by indigenous people to treat various ailments, including malaria, have been described. The most often used ethnobotanical plant species for the treatment of malaria were Allium sativum, Croton macrostachyus, Carica papaya, and Lepidium sativum. Leaves were used more frequently as a therapeutic preparation than other parts. The anti-malarial activity of the species investigated, as well as their potential as sources of new anti-malarial compounds and toxicities, is reviewed here. The most active species were Ajuga remota, Capsicum frufescens, Hagenia abyssinica, Zehenria scabra and Aloe macrocapa, which suppressed parasitaemia by 77 at an oral dose of 100 mg/kg and an LD 50 of above 2000 mg/kg. These are herbs that have traditionally been used to treat malaria. The compound Aloinoside (17) reported from Aloe macrocarpa leave latex and evaluated for anti-malarial activity; it suppressed parasitaemia by 100% at 400 mg/kg oral dose of P. berghei infected mice, and its LD 50 was above 2000 mg/kg. This suggests that this compound could be employed as an anti-malarial drug. Malaria control efforts and resources have expanded in Ethiopia, where the burden of malaria is the highest due to the country's vast population and geographical setting. In the light of these facts, this review focuses on Ethiopian medicinal plants used to treat malaria, as well as compounds purified from them, in the hope of helping eliminate the disease. Because it is hoped that the discovery of active compounds in plants would lead to the development of more effective drugs that are both economical and accessible to rural communities at the greatest risk of disease morbidity. However, no further investigation of the efficacy of several plant species that have been described as anti-malarial could be found. More studies are needed to identify and develop successful novel drugs that could be used in broader malaria eradication efforts.